The present invention relates to a method for determining theophylline in a sample. More particularly, the present invention relates to a method for determining theophylline which uses a theophylline utilizing enzyme.
Theophylline is a bronchodilator and respiratory stimulant used in the treatment of patients with asthmatic and allergic conditions. It is also used in the treatment of congestive heart failure and acute pulmonary edema. Benefits, as well as risks, from using this drug directly relate to its serum concentration. In order for the drug to be effective, a concentration of theophylline of about 10-20 mg/L level needs to be maintained in the blood. Theophylline levels of less than 10 mg/L are therapeutically ineffective and levels of more than 20 mg/L may be toxic to the patient. This toxicity may result in brain damage and death. Because the therapeutic advantage of the drug lies only within a narrow range of concentrations and because there is a large interpatient difference in drug elimination due to physiological differences, as well as diet or other prescribed drugs, it is important to monitor patients using this drug.
Theophylline has been measured by gas chromatography by Shah, J. Pharm. Sci., 63(8), 1283 (1974) and by a combination of gas chromatography and mass-selective detector by Desage, et al., J. Chromat., 336(2), 285 (1984). It has been measured by high-pressure liquid chromatography by Thompson, et al., J. Lab. Clin. Med., 84(4), 584 (1974) and by Naish, et al., Ann. Clin. Biochem., 16(5), 254 (1979). Schack, et al., J. Pharm., 97, 283 (1949) used an ultraviolet spectrophotometric method for the determination of theophylline. However, because these methods need cumbersome extractions, most clinical approaches today for the determination of theophylline use immunological methods which depend on antibodies for recognizing theophylline in the sample being tested. Such systems involve competitive protein binding where the antibody is the specific binding protein. After the reaction with antibodies takes place, the determination of theophylline varies depending on the particular assay, that is, the assay readout may be turbidimetric, nephelometric, radioactive or colorimetric depending on whether turbidity, radioactivity or color is produced. Examples of these systems are reported by Painter, et al., J. Clin. Lab. Autom., 3(3), 179 (1983), Samoszuk, et al., Ther. Drug Mon., 5(1), 113 (1983), Opheim, et al., Clin. Chem., 30(11), 1870 (1984), Boeckx and Munson, Ther. Drug Mon., 7(1), 95 (1985), Cook, et al., Res. Comm. in Chem. Path. & Pharm., 13(3), 497 (1976), and Landesman, et al., Clin. Chem., 29, 1238 (1983).
Immunological systems have also been reported by Li et al., Clin. Chem., 27(1), 22 (1981), Davis and Marks, Ther. Drug Mon., 5(4), 479 (1983), Chang, et al., Clin Chem 28(2), 361 (1982), Hinds, et al., Clin. Chem., 30(7), 1174 (1984), Jolley, et al., Clin. Chem., 27, 1575 (1983), Morris, et al., Anal. Chem., 53, 658 (1981), and Tyhach, et al., Clin. Chem., 27, 1499 (1981).
In addition, enzymes have been used in an enzyme amplification assay, U.S. Pat. No. 3,817,837. In this disclosure, enzymes are chemically bound to ligands and these enzyme-bound-ligands combine with receptors. The ligand may be a drug. The specific reaction of the ligand with the receptors gives the specificity to the reaction while the enzyme activity is utilized as a marker for the reaction. Therefore, the enzymes used have no enzymatic recognition of the drug. The use of these approaches are totally different to the presently described methodology which uses enzymes instead of antibodies or ligands for the recognition of theophylline.
It has been also reported in European Patent Application Number EP86300226.7, Jan. 15, 1986, as well as in Clin. Chem., 25, 1370 (1979), that the activity of alkaline phosphatase, which acts by cleaving phosphate groups from a substrate, can be inhibited by theophylline. In this approach, the enzymatic reaction of alkaline phosphatase continues to be that of cleaving the phosphate bonds but this action is interfered with by the presence of theophylline. Again, in this disclosure, the theophylline test produced is one in which theophylline is not enzymatically utilized or changed by the enzymatic reaction. By contrast, the present invention teaches that test systems can be produced, using enzymes which utilize or recognize theophylline and use it as a substrate for the quantitation of theophylline in samples.
Since theophylline is a xanthine derivative, commercially available xanthine oxidases and xanthine dehydrogenases and related enzymes were tried for their ability to utilize theophylline and to develop a test system. These attempts were unsuccessful. Although in humans, theophylline is known to be metabolized primarily to 1,3 dimethyluric acid and also to 1 methyluric acid and 3-methylxanthine (Cornish, H. H. and Christman, A. A., J. Biol. Chem., 228, 315 (1957), no theophylline utilizing enzyme has previously been isolated nor its use shown in a test system to quantitate theophylline in a sample.
Here, the present invention contemplates the measurement or quantitation of theophylline concentration using these theophylline utilizing or recognizing enzymes. Examples of these enzymes, namely, theophylline dehydrogenase, theophylline oxidase, and theophylline demethylase are used to demonstrate the efficacy of the method, test composition and test device of the present invention for the measurement of theophylline in samples such as body fluids, food extracts, and medicinal compounds and compositions. The tests that resulted from this enzymatic approach are rapid and convenient to perform. The advantages of the enzymatic approaches are 1) unitized reagent or test composition capability, 2) one step addition of sample to reagent or test composition, 3) a liquid system can be made to perform with instrument readout devices, and 4) the reagent or test composition can easily be incorporated into a solid-phase matrix. The present invention also contemplates a process of obtaining theophylline utilizing or recognizing enzymes from microbial sources which react with theophylline as a substrate and produce a product.